Method of preparing bilirubin standardized reagents



1956 T. E. WEICHSELBAUM 2,770,601

METHOD OF PREPARING BILIRUBIN STANDARDIZEID REAGENTS Filed Oct. 15. 1953 FIG.2

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M W L E S H m E W E a o D o E H T BY H013 @W ATTORNEY States METHOD OF PREPARING BILIRUBIN STANDARDIZED REAGENTS Theodore E. Weichselbaum, Normandy, Mo., assignor to A. S. Aloe Company, St. Louis, Mo., a corporation of Missouri 4 Claims. (Cl. 252-408) This invention relates to certain new and useful improvements in methods of preparing quantitative laboratory standards of the type used in clinical chemistry for verifying and correcting instrument calibration tables and graphs. This application is a divisional of my copending application, Serial No. 233,465, filed June 25, 1951, which, in turn, was a continuation in part of application Serial No. 778,681, filed October 8, 1947, and abandoned. My said application Serial No. 233,465 is now Patent No. 2,664,403, granted December 29, 1953.

At the present time, in the performance of many routine analyses, clinical laboratories make use of physical colorimetry and spectrophotometry as well as the practice of various related technics, such as those requiring the employment of photoelectric instruments and those involving titrimetry. Analyses of this character have exceedingly broad application and in the physiological field, for diagnostic purposes, are extensively used in the analysis of body fluids such as blood serum and plasma, urine, and spinal fluid. Such procedures, although routine or research in nature, normally require painstaking and laborious preliminary steps, including the exacting preparation of quantitative standards for color comparison purposes and for checking the accuracy of the instruments utilized so that any deviations in the operation thereof will be precisely adjusted for by calibration. Customarily, whatever excess may remain of these standards after the accomplishment of the color comparision or the instrument verification or correction procedure will usually be discarded since same are subject to deterioration, inadver tent dilution or contamination and, thus, their further use at a later date will be productive of inaccuracy. Consequently, it is necessary to prepare such standards immediately prior to their use. This procedure is manifestly inherently uneconomical and, furthermore, demands an element of proficiency on the part of the operator in preparing the standard since any errors in the formation thereof will perforce cause the determination of false results.

Furthermore, the preparation of many of such standards are most intricate and thereby demand marked skill and wide experience on the part of the technician. It is evident that the amount of time spent by skilled personnel in preparing such reagents constitutes a severe lack of economy of such individuals time since it diverts them from their immediate research pursuits and thereby causes devotion of a considerable proportion of their time on merely ancillary preparatory steps. With the developing shortage of highly trained scientific personnel, it is readily recognized that their efforts should at all times be directed solely to the problem at hand and not dissipated on preparatory procedures.

Therefore, it is a primary object of the present invention to provide a method of preparing quantitative laboratory standards incorporating the preparation of a dried, solid reagent of predetermined weight which may be dissolved in a selected diluent to form a standard of pre- 2,770,601 Patented Nov. 13, 1956 determined concentration suitable for accurate quantitative measurement.

It is a further object of the present invention to provide a method for preparing quantitative laboratory standards which comprises the desiccation of a predetermined quantity of a solution of known concentration and the maintenance of such desiccated portion under conditions wherein it will retain its properties indefinitely.

It is an additional object of the present invention to provide a method for preparing quantitative laboratory standards Which may be accurately and reliably performed by relatively unskilled operators.

It is a still further object of the present invention to provide a method of preparing quantitative laboratory standards which is highly economical, simple in performance, and convenient.

With the above and other objects in view, my inven- :tion resides in the novel methods and precesses presently described and pointed out int-he claims.

In the accompanying drawing- Figure 1 is a side elvational view of a preferred form of reagent ampule for use in the performance of the method of the present invention;

Figure 2 is a vertical cross-sectional view of the ampule shown in Figure 2; and

Figure 3 is a horizontal sectional view taken along line 3-3 of Figure l.

The method herein comprises the formation of a solution of desired concentration; the measuring of a predetermined quantity of the prepared solution and the depositing of the same within an ampule or other enclosable container; the dehydation or desiccation of the measured quantity in the ampule by any suitable means, such as by evaporation or by lyophilization, as in the manner set forth in United States Letters Patent No. 2,225,774. issued to E. W. Flosdorf, December 24, 1940; the sealing of the ampule under vacuum; and the ultimate utilization of the wholly dried, solid reagent by rupture of the amp ule and dissolution of the dried reagent in a predetermined quantity of selected diluent to reconstitute a quantitative standard of known concentration.

Referring now in more detail, and by reference characters to the drawing, which illustrates a practical type of ampule for use in the method herein taught, but is not a part of the present invention, A designates a blown or drawn glass ampule of essentially tubular form and integrally comprising a lower or body portion 1, an intermediate or constricted portion 2, and an upper portion 3 preferably of substantially the same diametral size as the body portion 1, tapering at its upper end into an axially upwardly projecting sealing neck 4, the latter being initially open for filling purposes and, subsequent to filling, being sealed, as at 5, in a hot flame, such as a Bunsen burner or blow lamp. The constricted portion 2 is provided, midway of its length, with a line of weakness or scratch line 6, and a small outwardly blown or drawn nipple-like protuberance 7, the apex or point of which is concident with the scratch line 6.

In the performance of the method of this invention, the reagent ampule A is filled with an accurately measured or weighed quantity of reagent having a precisely and the contents ofthe ampule desiccated by freezing the liquid contents and conductingdesiccation by sublimation.

various types of biological reagents which must be handled very carefully to avoid impairment of their action. After the product has been completely desiccated, thesealing neck 4 is sealed off and the product is completed, ready for labeling and final use. The precise conditions of such dehydration methods depend upon the character of the particular standard being prepared as some will require low temperature desiccation whereas others are reduced to a dry state by high temperature vacuum procedures.

In some cases, such as with a bilirubin standard, lyophilization is not used but the dehydration process is carried out by evaporation procedure with sealing of the desiccated material in an inert atmosphere, as will presently be more fully described. The manner of desiccation is thus dependent upon the particular substances involved.

Reagents so ampulized in wholly desiccated state will keep for indefinite periods without deterioration, remaining stable as to their original properties, so that an operator may use same, as will be shown below, whenever the particular occasion arises without fear of any inaccuracy resulting through untoward impairment of the reagent.

When the operator desires to make a quantitative laboratory standard for clinical analytical purposes, the ampule A is manually broken along the scratch line 6 into two sections, and the contents washed by a small quantity of the indicated diluent into the container in which such standard is to be made. It is to be particularly noted that exact quantitative transfer of the ampule con tents must be made. Therefore, the desiccated material in both of the sections of the now broken ampule must be completely dissolved and transferred. The rupturing of the ampule A along the scratch line 6 will result in the formation of a pouring lip to facilitate transference of the dissolved reagents into the container in which the standard is being made. Successive washings from a conventional laboratory wash bottle, or the like may be done to assure that all desiccated material has been totally dissolved and transferred. The solution is then made up to the predetermined or stated volume.

The quantitative standards so prepared by the present invention are particularly fitted for utilization in verifying the calibration tables and graphs of photoelectric colorimeters and spectrophotometers, as well as providing color standards for visual colorimetry and for titri'rnetric technics. As illustrative of the method herein described, below are presentedtwo specific examples of the preparation of a quantitative standard for checking the Cit accuracy of the type of calibration table supplied with a' I pre-calibrated spectrophotometer or of .a transmittanceconcentration graph. 7

Example I 13.88 mg, is deposited into the ampule A through the now open sealing neck 4. The 5 cc. of reagent is thensubjected to lyophilization which comprises shell freezing by immersion of the ampule'A into a suitable bath, such as isopropyl, alcohol at "4() C.-- Subsequent to the freezing'step, andbefore thawing can occur, a high vacuum is applied in order to evacuate all water thereby' leaving the reagent in a completely desiccated, solid from. The ampule A is then sealed under vacuum.

To reconstitute the reagent for analytical use, the ampule A is broken along the line of weakness 6, and smallamounts of distilled water, in-the neighborhoodof -1'0 ml., are deposited in the upper and lower portions 1, 3,

4 of the ampule A for dissolving the solid reagent. Thereon the portions 1, 3, are emptied into a 250 cc. flask. However, additional flushings of the ampule A may be made to assure quantitative dissolution of the entire reagent. The flask is then made up to the mark with the diluent to constitute a standard of accurate known strength, suitable for use as a standard in visual colorimetry and for use as a standard for calibration of verification of photoelectric and spectrophotometric curves or calibration tables. As a verification standard, it has a transmittance equivalent to 18.7 grams percent hemoglobin.

Example 11 To prepare protein standards for use in determining the total protein and albumin content of blootLiplasma is extracted from whole beef blood and the protein content thereof discovered 'by conventional technics. The beef plasma is then diluted with a saline solution, namely, .85 percent sodium chloride, in requisite amount to provide 360 mg. of protein per 5 cc. Then 5 cc. of the reagent thus formed is deposited in the ampule A and subjected to shell freezing and desiccation, after which the ampule A is sealed under vacuum. The solid reagent within the ampule A represents 9 grams percent total serum protein equivalent. To reconstitute the reagent, the ampule A is broken and flushed repeatedly with small amounts of the diluent, which in this case is 30 percent urea-thymol reagent, until the reagent is completely quantitatively dissolved, with transference being made to a ml. volumetric flask. The diluent is then added to the flask to make up to. the mark. Various concentrations of protein standard reagents may be made by this method, such as, one containing 280 mg. per 100 ml. which represents 7 grams percent total protein material or one of the concentration of mg. per 100 ml., which represents 4 grams percent total serum protein equivalent, and being suitable for use in color-imetry and for calibration for verification of photoelectric and spectrophotometric curves or calibration tables.

Thus, by standards ofthe type above set forth, a technician may accordingly verify or correct calibration tables to assure-accurate quantitative measurement. 7

The preparation of a bilirubin standard for use in quantitatively determining the presence of this bile pigment in serum or plasma, will serve as an example of the preparation of a reagent by evaporation. In this instance, a solution is made by dissolving 20 mg. bilirubin in 100 cc. of chloroform. Then 5 cc. of the reagent thus formed is deposited in the ampule A and subjected to evaporation to provide 17mg. of bilirubin in a dry, solid state, after which the ampule A is sealed in an inert atmosphere, with nitrogen being the gas used. Thus, the air in the ampulealcohol is then added to the flask to make up to the mark;

As described he-reinabove, it isunderstood that bothsections of the broken ampule may be repeatedly washed to assure exact, and precise' dissolution of all bilirubin.

The total amount of diluent utilized is roughly 95 percent methyl alcohol and 5 percent by volume chloroform.

Thesolution so formed represents 1mg. percent bilirubin equivalent .and will accordingly provide accurate light transmittance for such concentration to serve as a reliable working standard.

It should be understood that the present invention resides in the unique method of providing dry,.s0lid re- 7 agents of known strength from a solution of predetermined concentration, which reagent'is adapted for reconstituting a solution of the identical concentration at a subsequent time for quantitative measurement purposes in chemical and clinical analysis.

It is, of course, apparent that the present method is equally designed for use by physicians, analytical chemists, and laboratory technicians in both the medical and industrial fields, and obviously does not require developed skill on the part of the operator. Thus, relatively untrained assistants may perform the method of the present invention without substantial hazard of preparing an incorrect standard. The economy in material, time, and labor implicit in the practice of the method is apparent.

It should be understood that changes and modifications in the methods above set forth and in the various steps of their production may be made and substituted for those herein shown and described without departing from the nature and principle of the present invention.

Having thus described my invention, what I claim and desire to secure by Letters Patent is:

1. The method of making bilirubin standardized reagents for use in colorime-try and in checking the accuracy of spectrophotometric calibration tables which method comprises providing a predetermined quantity of bilirubin, dissolving the measured quantity of bilirubin in a predetermined volume of chloroform to provide a solution of predetermined concentration, placing a measured quantity of solution within a glass container, evaporating the liquid material in the container for reduction thereof to a dry, solid state, then sealing the container in an inert atmosphere, opening the container at the time of preparation of the standard, dissolving the dry reagent in such amount of chloroform as requisite for complete dissolution of the dried material, then quantitatively transferring same by methyl alcohol to a flask having a volume-indicating mark, and then making up to the mark with methyl alcohol to constitute a standard of predetermined concentration.

2. The method of making bilirubin standardized reagents for use in colorimetry and in checking the accuracy of spectrophotometric calibration tables which method comprises providing a predetermined quantity of bilirubin, dissolving the measured quantity of bilirubin in a predetermined volume of a solvent to provide a solution of predetermined concentration, placing a measured quantity of solution within a glass container, evaporating the liquid material in the container for reduction thereof to a dry, solid state, then sealing the container in an inert atmosphere, opening the container at the time of preparation of the standard, dissolving the dry reagent 'in such amount of a solvent as requisite for complete dissolution of the dried material, then quantitatively transferring same by methyl alcohol to a flask having a volume-indicating mark, and then making up to the mark With methyl alcohol to constitute a standard of predetermined concentration.

3. The method of making bilirubin standardized reagents for use in colorimetry and in checking the accuracy of spectrophotometric calibration tables which method comprises providing a predetermined quantity of bilirubin, dissolving the measured quantity of bilirubin in a predetermined volume of chloroform to provide a solution of predetermined concentration, placing a measured quantity of solution within a glass container, evaporating the liquid material in the container for reduction thereof to a dry, solid state, then sealing the container in an inert atmosphere, opening the container at the time of preparation of the standard, dissolving the dry reagent in such amount of chloroform as requisite for complete dissolution of the dried material, then quantitatively transferring same by methyl alcohol to a flask having a volume-indicating mark, and then making up to the mark with methyl alcohol to constitute a standard of predetermined concentration.

4. The method of making bilirubin standardized reagents for use in colon'metry and in checking the accuracy of spectrophotometric calibration tables which method comprises providing a predetermined quantity of bilirubin, dissolving the measured quantity of bilirubin in a predetermined volume of chloroform to provide a solution of predetermined concentration, placing a measured quantity of solution within a glass container, evaporating the liquid material in the container for reduction thereof to a dry, solid state, then sealing the container in an inert atmosphere, opening the container at the time of preparation of the standard, dissolving the dry reagent in such amount of chloroform as requisite for complete dissolution of the dried material, then quantitatively transferring same by methyl alcohol to a flask having a volume-indicating mark, and then making up to the mark with methyl alcohol to constitute a standard of predetermined concentration, wherein the diluent is approximately by volume 5% chloroform and methyl alcohol.

References Cited in the file of this patent UNITED STATES PATENTS 2,085,391 Reichel June 29, 193'? 

1. THE METHOD OF MAKING BILIRUBIN STANDARDIZED REAGENTS FOR USE IN COLORIMETRY AND IN CHECKING THE ACCURACY OF SPECTROPHOTOMETRIC CALIBRATION TABLES WHICH METHOD COMPRISES PROVODING A PREDETERMINED QUANTITY OF BILIRUBIN, DISSOLVING THE MEASURED QUANTITY OF BILIRUBIN IN A PREDETERMINED VOLUME OF CHLOROFORM TO PROVIDE A SOLUTION OF PREDETERMINED CONCENTRATION, PLACING A MEASURED QUANTITY OF SOLUTION WITHIN A GLASS CONTAINER, EVAPORATING THE LIQUID MATERIAL IN THE CONTAINER FOR REDUCTION THEREOF TO A DRY, SOLID STATE, THEN SEALING THE CONTAINER IN AN INERT ATMOSPHERE, OPENING THE CONTAINER AT THE TIME OF PREPARATION OF THE STANDARD, DISSOLVING THE DRY REAGENT IN SUCH AMOUNT OF CHLOROFORM AS REQUISITE FOR COMPLETE DISSOLUTION OF THE DRIED MATERIAL, THEN QUANTITATIVELY TRANSFERRING SAME BY METHYL ALCOHOL TO A FLASK HAVING A VOLUME-INDICATING MARK, AND THEN MAKING UP TO THE MARK WITH METHYL ALCOHOL TO CONSTITUTE A STANDARD OF PREDETERMINED CONCENTRATION. 